sequence reads are aligned to a reference genome. Following alignment, many variations of
analysis are possible, such as single nucleotide polymorphism (SNP) or insertion-deletion
(indel) identification, read counting for RNA methods, phylogenetic or metagenomic
analysis, and more. A graphical overview of the NGS chemistry is depicted in Fig. 4.1.
4.3
Ion Torrent
Unlike Illumina Ion Torrent semiconductor sequencing from Thermo Fisher Scientific does
not make use of optical signals. Instead, they exploit the fact that addition of a dNTP to a
DNA polymer releases an H
+ ion.
As in other kinds of NGS, the input DNA or RNA is fragmented to approximately
200bp, adapters are added, and one molecule is placed onto a bead. The molecules are
amplified on the bead by emulsion PCR resulting in millions of different beads with
millions of different fragments. These beads than flow across the semiconductor chip
depositing each bead into a single well. Next the slide is flooded with a single species of
dNTP (one NTP at a time), along with buffers and polymerase. The pH is detected, as each
H
+ ion released will decrease the pH. The changes in pH allow to determine if that base,
and how many thereof, was added to the sequence read. The dNTPs are washed away and
the process is repeated cycling through the different dNTP species. The pH change (if any)
is utilized to determine how many bases (if any) were added with each cycle.
If a nucleotide, for example, a C, is added to a DNA template and is then incorporated
into a strand of DNA, a hydrogen ion will be released. The charge from that ion will change
the pH of the solution in the well, which can be detected by a specific ion sensor. This
process happens simultaneously in millions of wells, that is why this technology is often
described as massively parallel sequencing.
The Ion Torrent NGS instruments Genexus, Ion GeneStudio S5, ION PGM Dx, Ion
Chef, and Ion OneTouch2 are essentially the world’s smallest solid-state pH meters, calling
the base, going directly from chemical information to digital information.
4.4
Pacific Bioscience
Single-molecule, real-time (SMRT) sequencing developed by Pacific BioSciences
(PacBio) offers longer read lengths than the second-generation sequencing (SGS)
technologies, making it well-suited for unsolved problems in genome, transcriptome, and
epigenetics research [5].
Introducing the PacBio Sequel II system powered by SMRT sequencing technology the
first step is to isolate DNA or RNA from any sample type. Next a SMRTbell library is
created by ligating hairpin adapters to double stranded DNA creating a circular template.
Primer and polymerase are added to the library that is placed on the instrument for
sequencing. The smart cell contains millions of small, tiny wells called zero-mode
waveguides (ZMWs). A single molecule of DNA is immobilized in a ZMW sequencing
4 NGS Technologies
51
analysis are possible, such as single nucleotide polymorphism (SNP) or insertion-deletion
(indel) identification, read counting for RNA methods, phylogenetic or metagenomic
analysis, and more. A graphical overview of the NGS chemistry is depicted in Fig. 4.1.
4.3
Ion Torrent
Unlike Illumina Ion Torrent semiconductor sequencing from Thermo Fisher Scientific does
not make use of optical signals. Instead, they exploit the fact that addition of a dNTP to a
DNA polymer releases an H
+ ion.
As in other kinds of NGS, the input DNA or RNA is fragmented to approximately
200bp, adapters are added, and one molecule is placed onto a bead. The molecules are
amplified on the bead by emulsion PCR resulting in millions of different beads with
millions of different fragments. These beads than flow across the semiconductor chip
depositing each bead into a single well. Next the slide is flooded with a single species of
dNTP (one NTP at a time), along with buffers and polymerase. The pH is detected, as each
H
+ ion released will decrease the pH. The changes in pH allow to determine if that base,
and how many thereof, was added to the sequence read. The dNTPs are washed away and
the process is repeated cycling through the different dNTP species. The pH change (if any)
is utilized to determine how many bases (if any) were added with each cycle.
If a nucleotide, for example, a C, is added to a DNA template and is then incorporated
into a strand of DNA, a hydrogen ion will be released. The charge from that ion will change
the pH of the solution in the well, which can be detected by a specific ion sensor. This
process happens simultaneously in millions of wells, that is why this technology is often
described as massively parallel sequencing.
The Ion Torrent NGS instruments Genexus, Ion GeneStudio S5, ION PGM Dx, Ion
Chef, and Ion OneTouch2 are essentially the world’s smallest solid-state pH meters, calling
the base, going directly from chemical information to digital information.
4.4
Pacific Bioscience
Single-molecule, real-time (SMRT) sequencing developed by Pacific BioSciences
(PacBio) offers longer read lengths than the second-generation sequencing (SGS)
technologies, making it well-suited for unsolved problems in genome, transcriptome, and
epigenetics research [5].
Introducing the PacBio Sequel II system powered by SMRT sequencing technology the
first step is to isolate DNA or RNA from any sample type. Next a SMRTbell library is
created by ligating hairpin adapters to double stranded DNA creating a circular template.
Primer and polymerase are added to the library that is placed on the instrument for
sequencing. The smart cell contains millions of small, tiny wells called zero-mode
waveguides (ZMWs). A single molecule of DNA is immobilized in a ZMW sequencing
4 NGS Technologies
51